A major contributing factor to the high mortality rate associated with acute myeloid leukemia (AML) is the development of resistance to chemotherapy and death receptor-mediated apoptosis. Multiple myeloma (MM) is one of the most common hematological malignancies, involving bone marrow plasmacytosis. Multiple myeloma (MM) is currently incurable, with approximately 20,000 newly diagnosed cases per year and the current median survival is 3-5 years. Uncontrolled proliferation of plasma cells in the bone marrow ultimately leads to hematopoietic failure and skeletal destruction. Oncogene mutations, translocations in immunoglobulin enhancers, and cytogenetic changes result in progression from a germinal center B cell to multiple myeloma, which occurs in approximately 1%-3% of the adult population (Dalton W S, et al. (2001) Multiple myeloma. Hematology Am Soc Hematol Educ Program: 157-177).
Invasion of the bone marrow by the malignant plasma cell neoplasm leads to hematopoietic failure, clotting via thrombocytopenia, anemia via diminished erythrocyte production and survival (Silvestris F, et al. (2002) Upregulation of erythroblast apoptosis by malignant plasma cells: a new pathogenetic mechanism of anemia in multiple myeloma. Rev Clin Exp Hematol Suppl 1:39-46; Silvestris F, et al. (2002) Negative regulation of erythroblast maturation by Fas-L(+)/TRAIL(+) highly malignant plasma cells: a major pathogenetic mechanism of anemia in multiple myeloma. Blood 99:1305-1313; Pratt G, et al. (2007) Immunodeficiency and immunotherapy in multiple myeloma. Br J Haematol 138:563-579), and destruction of the skeleton through enhanced osteoclast activity and suppressing osteoblast differentiation (Lentzsch S, et al. (2007) Pathophysiology of multiple myeloma bone disease. Hematol Oncol Clin North Am 21:1035-1049, viii). Other complications of the disease, such as renal failure (Gu X and Herrera G A (2006) Light-chain-mediated acute tubular interstitial nephritis: a poorly recognized pattern of renal disease in patients with plasma cell dyscrasia. Arch Pathol Lab Med 130:165-169), also contribute to morbidity and mortality in the patients. MM is associated with a poor prognosis relative to lymphomas and leukemias in general, with a median life expectancy of 3-4 years (U.S. Government Statistics N (2009) Surveillance Epidemiology and End Results, in (Institute NC ed)).
There are currently various treatment options for the management of MM, influenced by various factors including patient age and eligibility for autologous stem cell transplantation (ASCT). Treatments include high-dose dexamethasone-based regimens, melphalan (Facon T, et al. (2006) Dexamethasone-based regimens versus melphalan-prednisone for elderly multiple myeloma patients ineligible for high-dose therapy. Blood 107:1292-1298), combinations of vincristine, doxorubicin (Adriamycin) and dexamethasone (Decadron) (Reece DE (2007) Management of multiple myeloma: the changing landscape. Blood Rev 21:301-314), thalidomide-like drugs (i.e. thalidomide and lenalidomide) and proteosome inhibitors (i.e. bortezomib). While some of these regimens have produced higher initial response rates, this has not translated into improved overall survival outcomes (Ludwig H, et al. (2009) Thalidomide-dexamethasone compared with melphalan-prednisolone in elderly patients with multiple myeloma. Blood 113:3435-3442). Thus, there is a significant need to develop novel therapeutic options that improve outcomes and extend survival. Surprisingly, the rate of relapse is consistent for both the various treatment regimens and initial response to treatment.
Cancer cells frequently evade chemotherapy-induced cell death via a process referred to as de novo drug resistance, which is likely one of the first steps involved in acquired drug resistance (Nefedova Y, et al. (2003) Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms. Leukemia 17:1175-1182). The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and soluble factors including cytokines and growth factors. All of these components interact with tumor cells to contribute to de novo drug resistance (Li Z W and Dalton W S (2006) Tumor microenvironment and drug resistance in hematologic malignancies. Blood Rev 20:333-342), and the persistence of minimal residual disease with currently available drug regiments. This minimal residual disease ultimately permits relapse, regardless of the initial observable response. Of particular interest in this process is the ECM protein, fibronectin, (Hazlehurst L A and Dalton W S (2001) Mechanisms associated with cell adhesion mediated drug resistance (CAM-DR) in hematopoietic malignancies. Cancer Metastasis Rev 20:43-50; Hazlehurst L A, et al. (2000) Adhesion to fibronectin via beta1 integrins regulates p27kip1 levels and contributes to cell adhesion mediated drug resistance (CAM-DR). Oncogene 19:4319-4327). The adhesion of tumor cells to fibronectin cells and bone marrow stromal cells has been implicated in the cellular rearrangement of molecules involved in drug resistance. This particular form of de novo drug resistance is called cell adhesion-mediated drug resistance (CAM-DR), and the β1 integrin subunit appears to be a key player in development of this resistance.
Farnesyltransferase inhibitors are a novel class of anticancer agents developed to inhibit the enzyme farnesyltransferase that is responsible for the transfer of a farnesyl group to the Ras protein. FTIs were originally designed to inhibit Ras oncogenic activity, but recent studies suggest that FTIs may have several other targets including centromeric proteins and the phosphatidylinositide-3 kinase/Akt pathway (Ashar H R, et al. Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules. J Biol. Chem. 2000 Sep. 29; 275(39):30451-7; Jiang K, et al. The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol. 2000 January; 20(1):139-48). To date, several FTIs have been clinically evaluated, including BMS-214664, SCH-66363 and R115777 for myelodysplastic syndrome, chronic myelogenous leukemia and acute leukemias (Johnston S R, Kelland L R. Farnesyl transferase inhibitors—a novel therapy for breast cancer. Endocr Relat Cancer. 2001 September; 8(3):227-35; Karp J E, et al. Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial. Blood. 2001 Jun. 1; 97(11):3361-9; Kurzrock R, et al. Farnesyltransferase inhibitor R115777 in myelodysplastic syndrome: clinical and biologic activities in the phase 1 setting. Blood. 2003 Dec. 15; 102(13):4527-34). Tipifarnib (R115777) is a potent non-peptidomimetic inhibitor of farnesyltransferase (Kohl N E. Farnesyltransferase inhibitors. Preclinical development. Ann N Y Acad. Sci. 1999; 886:91-102; Santucci R, et al. Farnesyltransferase inhibitors and their role in the treatment of multiple myeloma. Cancer Control. 2003 September-October; 10(5):384-7; Sebti S M, Adjei A A. Farnesyltransferase inhibitors. Semin Oncol. 2004 February; 31(1 Suppl 1):28-39). Studies have demonstrated the anticancer activity of tipifarnib as a single agent or in combination in preclinical models for multiple myeloma and acute myeloid leukemia (Beaupre D M, et al. Farnesyl transferase inhibitors enhance death receptor signals and induce apoptosis in multiple myeloma cells. Leuk Lymphoma. 2003 December; 44(12):2123-34; Alsina M, et al. Farnesyltransferase inhibitor tipifarnib is well tolerated, induces stabilization of disease, and inhibits farnesylation and oncogenic/tumor survival pathways in patients with advanced multiple myeloma. Blood. 2004 May 1; 103(9):3271-7; Yanamandra N, et al. Tipifarnib and bortezomib are synergistic and overcome cell adhesion-mediated drug resistance in multiple myeloma and acute myeloid leukemia. Clin Cancer Res. 2006 Jan. 15; 12(2):591-9; Zhu K, et al. Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells. Blood. 2005 Jun. 15; 105(12):4759-66; Beaupre D M, et al. R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways. Mol Cancer Ther. 2004 February; 3(2):179-86). However, the molecular mechanisms by which tipifarnib triggers cell death still remain elusive, and has not been unequivocally associated with farnesyltransferase inhibition.
Programmed cell death, or apoptosis, is a genetically controlled and evolutionary conserved mechanism required for normal development and tissue homeostasis (Fadeel B, et al. Apoptosis in human disease: a new skin for the old ceremony? Biochem Biophys Res Commun. 1999 Dec. 29; 266(3):699-717). There are two main apoptotic pathways that trigger apoptosis and these are the extrinsic and intrinsic death pathways, respectively. The extrinsic pathway is triggered by the ligation of cell surface death receptors to death receptor ligands followed by formation of the death inducible signaling complex, which results in the activation of caspase-8 and subsequent downstream effectors (Peter M E, Krammer P H. Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis. Curr Opin Immunol. 1998 October; 10(5):545-51; Alnemri E S. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem. 1997 January; 64(1):33-42). The intrinsic cell death pathway is initiated by mitochondrial release of cytochrome C, resulting in formation of the apoptosome complex and activation of caspase-9 (Chinnaiyan A M. The apoptosome: heart and soul of the cell death machine. Neoplasia. 1999 April; 1(1):5-15). Recently, a third pathway has been identified that is initiated by the endoplasmic reticulum (ER), and is known as the ER-stress pathway (Lai E, et al. Endoplasmic reticulum stress: signaling the unfolded protein response. Physiology (Bethesda). 2007 June; 22:193-201).
Studies have shown that pathways involved in cell death and Ca2+ processing cross-talk, whereby activation of apoptotic cascades can interfere with the sequestration of Ca2+ into intracellular pools. Conversely, disruption in Ca2+ sequestration is sufficient to trigger apoptosis as a response to cellular stress (Orrenius S, et al. Regulation of cell death: the calcium-apoptosis link Nat Rev Mol Cell Biol. 2003 July; 4(7):552-65). Calcium storage and folding and sorting of newly synthesized proteins are the primary function of the mammalian ER. Imbalance in any of these processes can lead to ER stress, which in turn can activate at least two pathways. First, the unfolded protein response leads to induction of ER chaperons, such as GRP78, GRP94 and the transcription factor CHOP/GADD153 (Lai E, et al. Endoplasmic reticulum stress: signaling the unfolded protein response. Physiology (Bethesda). 2007 June; 22:193-201). Second, the ER overload response leads to activation of several signaling pathways including nuclear factor-κ-B (NF-κ-B) (Pahl H L, et al. Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention. J. Cell Biol. 1996 February; 132(4):511-22). Both responses help regulate the accumulation of misfolded proteins and enhance survival, but under severe stress they can also activate programmed cell death (Lee K, et al. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev. 2002 Feb. 15; 16(4):452-66).
Two basic components comprise environment-mediated drug resistance: physical contact between tumor cells and microenvironment components (cell adhesion mediated drug resistance, CAM-DR) and the local production of soluble factors. More specifically, in multiple myeloma and AML, it has been found that the adhesion of tumor cells (via integrin receptors) to fibronectin results in a drug-resistant phenotype (Hazlehurst L A, et al. Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death. Oncogene 2003; 22:7396-402; Matsunaga T, et al. Interaction between leukemic-cellVLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia. Nat Med 2003; 9:1158-65). Of importance, in a small series of AML patients, it was noted that those whose leukemic cells expressed VLA-4 (a4h1 integrin) had a high rate of relapse compared with those with low VLA-4 expression (Matsunaga T, et al. Interaction between leukemic-cellVLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia. Nat Med 2003; 9:1158-65). These results imply that the physical interaction between tumor cells and bone marrow constituents provides a refuge for minimal residual disease.
Many chemotherapeutic agents prompt apoptosis via the activation of one of these pathways. Cancer cells, however, frequently evade chemotherapy induced cell death via a process referred to as de novo drug resistance, which is likely one of the first steps involved in acquired drug resistance (Nefedova Y, et al. Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms. Leukemia. 2003 June; 17(6):1175-82). A major contributing factor to the high mortality rate associated with acute myeloid leukemia (AML) and multiple myeloma (MM) is the development of resistance to chemotherapy and death receptor-mediated apoptosis. The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and soluble factors including cytokines and growth factors. All of these components interact with tumor cells to contribute to de novo drug resistance (Li Z W, Dalton W S. Tumor microenvironment and drug resistance in hematologic malignancies. Blood Rev. 2006 November; 20(6):333-42). Of particular interest in this process is the ECM protein, fibronectin (Hazlehurst L A, et al. Reduction in drug-induced DNA double-strand breaks associated with beta1 integrin-mediated adhesion correlates with drug resistance in U937 cells. Blood. 2001 Sep. 15; 98(6):1897-903). Fibronectin is the ligand for at least ten integrin molecules that mediates cell adhesion (Li Z W, Dalton W S. Tumor microenvironment and drug resistance in hematologic malignancies. Blood Rev. 2006 November; 20(6):333-42). The adhesion of tumor cells to fibronectin and bone marrow stromal cells has been implicated in the cellular rearrangement of molecules involved in drug resistance including c-FLIP, Topoisomerase IIB, Fas and Bcl-2 (Hazlehurst L A, et al. Reduction in drug-induced DNA double-strand breaks associated with beta1 integrin-mediated adhesion correlates with drug resistance in U937 cells. Blood. 2001 Sep. 15; 98(6):1897-903). This particular form of de novo drug resistance is called cell adhesion-mediated drug resistance (CAM-DR). For example, in 15% of MM patient there is a t(4; 14) chromosomal translocation that results in overexpression of the tyrosine kinase receptor, fibroblast growth factor receptor 3 (FGFR3). Several studies have now shown that anti-FGFR3 antibodies are cytotoxic in t(4; 14)-positive multiple myeloma in mice and primary human t(4; 14)-positive MM cells (Trudel S, et al. (2006) The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t(4; 14) multiple myeloma cells. Blood 107:4039-4046; Qing J, et al. (2009) Antibody-based targeting of FGFR3 in bladder carcinoma and t(4; 14)-positive multiple myeloma in mice. J Clin Invest 119:1216-1229).
Multiple myeloma is an incurable hematologic malignancy that is the second most prevalent blood cancer after non-Hodgkin's lymphoma, affecting >56,000 people in the United States. While several new drugs have been identified in the last decade, and these treatments have increased the initial response rate; these new agents have failed to increase life expectancy. Moreover, these drugs have significant adverse effects that limit their usefulness. Thus, there is a great need for the identification of novel biomarkers and targets that will provide superior therapy for MM. Successful execution of the current proposal may reveal that Orai3 is such a biomarker/target, and provide a viable treatment alternative for a number of patients suffering from multiple myeloma. Tipifarnib would provide various treatment advantages over current drug regimens, including decreased toxicity, and may be quickly moved to clinical studies because of the substantial amount of human data already available for the drug.
Numerous proteins are involved in the movement of Ca2+ including ATPases, transporters and channels; and various intracellular compartments including mitochondria and the endoplasmic reticulum (ER) serve as buffers and stores for Ca2±. Dysregulation of intracellular Ca2±, which triggers protein misfolding in the ER, is linked to numerous responses including lipolysis, proteolysis, and oxidative stress. The accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress and activates the unfolded protein response (UPR) and the ER-associated degradation (ERAD). However, if the UPR is unable to resolve the underlying condition and ER stress becomes severe, programmed cell death is activated. Due to the ability of Ca2+ to lead cells in this pathway, there is a considerable amount of interest in targeting intracellular Ca2+ homeostasis for anticancer therapies.
Thus, there is a significant need to develop novel therapeutic options that improve outcomes and extend survival.